Processing of messenger RNA 3′ termination sequences resulting in polyadenylation is a universal feature of gene expression in eukaryotic organisms (see, for example, Nevins, J. R., “The pathway of eukaryotic mRNA formation”, Ann. Rev. Biochem. (1983) 52:441-466). This type of processing also has profound effects on gene expression, including total cessation of mRNA translation, as both mRNA stability and translatability are linked to polyadenylation. (Wickens, M., et al., “Life and Death in the Cytoplasm: Messages from the 3′ termination sequence”, Curr. Opin. Genet. Dev. (1997) 7:220-232). Evidence is accumulating that such alterations in 3′ termination sequence processing represents a form of expressional control which is directed by the interaction of trans-factors with cis-elements found in the precursor mRNA 3′ termination sequences.
Understanding the role of 3′ termination sequence processing in gene expression becomes critical when considering methods of expressing heterologous genes comprising “foreign” 3′ termination sequences. This is especially true in the case of plants where the introduction of foreign genes makes dramatic improvements in crop plants feasible through otherwise straightforward gene transfer technology. However, despite extensive research, attempts to express foreign genes with non-plant 3′ termination sequences in plants have thus far met with failure. For example, plant cells have been reported to be unable to recognize 3′ termination sequences in Saccharomyces cerevisiae genes (see, e.g., Barton, K. A., et al., Cell (1983) 32:1033-1043 and Irniger, S., et al., “Different Sequence Elements are required for function of Cauliflower Mosaic Virus Polyadenylation Site in Saccharomyces cerevisiae Compared with in Plants”, Mol. and Cell. Biol. (1992) 2322-2330), as well as many other sources (see, e.g., Koncz, C. et al., “A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and α-actin in plant tumors”, EMBO J. (1984) 3:(5):1029-1037).
This apparent lack of functionality of foreign 3′ termination sequences in plants has lead to a scarcity of 3′ termination sequences suitable for use in plant expression vectors for heterologous genes. In effect, only plant and plant viral 3′ termination sequences can currently be considered for use in such vectors and, of the possible functional 3′ termination sequences, only a few have been developed due to the difficulties in operably linking heterologous sequences to form a functional gene. Still other plant 3′ termination sequences are unsuitable as they lead to undesirable recombination events with native sequences or trigger “gene silencing” through various mechanisms such as the formation of anti-sense RNA species. This set of circumstances increases the complexity of expressing foreign genes in plant cells and severely limits a primary method of controlling genetic expression in response to tissue type, environmental stimuli, and other factors. Identification of non-plant 3′ termination sequences which are functional in plants, 3′ cis regulatory elements necessary for expression in plants, and methods for constructing novel 3′ termination sequences capable of functioning in plants would therefore be a significant advance in the expression of foreign genes in plant species.